This invention relates generally to oxygen determinations and more particularly has reference to methods and apparatuses for determining the concentration of oxygen in a gaseous or liquid environment based on luminscence quenching.
The determination of oxygen concentrations in gaseous samples, aqueous samples, and biological fluids has important ramifactions in medicinal, environmental and analytical chemistry. Today most oxygen measurements are based on modifications to the Clark electrode, Clark, Jr., L. C.; Trans. Am. Artif. Intern. Organs 1956, 2, 41, although the Winkler titration is also widely used, Skoog, D. A.; West, D. M.; Fundamentals of Analytical Chemistry, 3rd Edition, Holt, Rinehart and Winston, New York 1976, p. 369. The Clark electrode is easily calibrated, relatively rapid in response, and requires relatively inexpensive instrumentation. However, Clark electrodes consume oxygen and are easily poisoned by H.sub.2 S, proteins, and various organic compounds. In operating room use, they register large oxygen concentrations in the presence of certain anesthetics, which is a potentially fatal shortcoming, Albery, W. J.; Hahn, C. E. W.; Brooks, W. N.; Br. J. Anaesth. 1981, 53, 447.
The Winkler titration is slow and cumbersome. Further, since it is based on oxidation-reduction chemistry, interferences are numerous.
It is known that many platinum group metal complexes luminesce intensely in the red region (600-650 nm) when excited with visible light or UV light (&lt;550 nm). Both the intensity and the lifetime of the luminescence is decreased when the complex is exposed to deactivators (quenchers). Oxygen, iron(III), copper(II), and mercury(II) are among the common quenchers. When a single quencher is present in an environment, the degree of intensity or lifetime quenching is directly related to the quencher concentration and can be used as an analytical method for determining that concentration. However, the inability of the method to discriminate among different quenchers in an environment has herefore prevented the method from being universally applicable.
The discrimination problem is particularly acute when dealing with a liquid environment. If the luminescent complexes are dissolved directly in the solution, a variety of dissolved organic and inorganic contaminants and interferents contribute to the quenching and produce erroneous indications of oxygen concentrations.
Because the luminescence quenching method presents the possibility of making oxygen determinations without the limitations inherent in the Winkler titration method and the oxygen electrode method, it is desirable to improve upon known methods and apparatuses in the luminescence quenching art in order to make the method universally applicable.
The art is largely unpredictable because there are large environmental effects on luminescence behavior. The existence of emission and quenching in one matrix in no way guarantees the material will emit in another matrix. There are generally large changes in luminescent and quenching properties ongoing from solution to solid matrices, especially at the high concentrations needed by many sensors. It would be impossible to predict the suitability of materials in solid sensor matrices even for experts having more than 20 years of experience in this art.
Luminescence phase shifts are well known by practioners in the art. Phase shift measurements were suggested at least as early as 1904, and the first successful phase shift lifetime measurement was made by E. Gaviola in 1926 (Ann. Phys. (Leipzig) 81, 23 (1926)). For a full summary of phase shift lifetime measurement see the review by F. W. J. Teale in "Time Resolved Fluorescence Spectroscopy in Biochemistry and Biology" (Ed by R. B. Cundall and R. E. Dale, Plenum Press, 1983, p. 59).
The relating of quenching and quencher concentration dates back to the work of Stern and Volmer (1931). A recent biochemical example is taken from the work of J. R. Lakowicz and G. Weber (Biochemistry 12 4161 (1973)) who used the phase shift method to quantitate the degree of deactivation of a luminescent species by oxygen. This work was not used to measure oxygen concentrations and furthermore, the system as described would not be suitable for any such measurements.
Luminescence measurement analysis for monitoring concentrations of analytes is well known in the art. One merely makes a calibration curve of intensity verses concentration. When luminescent materials are excited by a modulated excitation source, the phase shift between the excitation beam and the emission beam can be used to determine the luminescent lifetime.
Pertinent United States and foreign patents are found in Class 23, subclasses 26, 52, 83, 230, 259, 906 and 927; Class 73, subclass 19; Class 204, subclasses 1, 1Y, 192P and 195; Class 250, subclasses 71 and 361C; Class 252, subclasses 18.3CL and 301.2; and Class 422, subclasses 52, 55-58, 83, 85-88 and 92 of the Official Classifications of Patents in the U.S. Patent and Trademark Office.
Examples of pertinent patents are U.S. Pat. Nos. 998,091; 1,456,964; 2,351,644; 2,929,687; 3,112,999; 3,697,226; 3,725,658; 3,764,269; 3,768,976; 3,881,869; 3,897,214; 3,976,451; 4,054,490; 4,073,623; 4,089,797; 4,181,501; 4,231,754; 4,260,392; 4,272,249; 4,272,484; and 4,272,485.
U.S. Pat. No. 3,725,658 shows a method and apparatus for detecting oxygen in a gas stream. The apparatus employs a sensor film comprising a fluorescent material dissolved in a carrier or solvent and supported on a substrate. Oxygen contained in the gas stream is dissolved into the film and quenches the fluorescent emission, the extent of quenching being proportional to the oxygen content of the gas stream. This patent claims four organic sensor materials, i.e., pyrene, coronene, p-terphenyl, and ovalene in addition to all fluorescent materials which absorb in the 2000-6500 .ANG. range and emit in the 3000-8000 .ANG. range. However, phosphorescent detectors are specifically excluded as potential sensor materials because of stability problems of the then known complexes. The stability problems were primarily centered on irreversibility of phosphorescing complexes. Moreover, emission intensity and not lifetime of quenching is the method of quantitation.
Stanley and Kropp, U.S. Pat. No. 3,725,658, explicitly use liquid or grease solvents as carriers. These include mineral oil, decalin, glycerol, and Apiezon N vaccum grease (column 3, lines 50-58). In the claims section they claim only a solvent " . . . selected from the class consisting of mineral oil, decalin, glycerol, tetrahydrofuran, benzene, and hexane." (column 8, lines 54-56). They also mention (column 4, lines 49-56) the protection of their sensor with plastic films when the sensor is absorbed onto silica gel, alumina, etc. This is quite different from incorporating the sensor directly into a polymer.
U.S. Pat. No. 3,764,269 shows the use of a gas permeable membrane which permits diffusion of a particular gas while providing protection against adverse effects of the environment. An electrochemical device detects the concentration of gas which passes through the porous layer and activates the electrode.
U.S. Pat. No. 3,881,869 discloses the chemiluminescent detection of ozone concentration in a gas sample. The gas sample contacts an organic polymer having a backbone chain consisting of carbon atoms to produce a chemiluminescent reaction. The concentration of ozone is proportional to the intensity of light emitted by the reaction.
U.S. Pat. No. 4,089,797 discloses chemiluminescent warning capsules having an air-reactive chemiluminescent formulation encapsulated with a catalyst. Crushing the capsule mixes the air-reactive formulation and the catalyst in the external environment to produce chemiluminescence if air is present.
U.S. Pat. No. 4,272,484 uses fluorescence methods to measure oxygen content after first separating blood protein fractions and other components by use of a gas permeable membrane. U.S. Pat. No. 4,272,485 is a related disclosure which includes a carrier which transports particles through the membrane.
U.S. Pat. No. 3,112,999 discloses a method where a gas, particularly carbon monoxide, permeates a porous layer to make an indication.
U.S. Pat. No. 2,929,687 discloses a dissolved oxygen test.
U.S. Pat. No. 3,768,976 shows a polymeric film through which oxygen migrates to cause an indication.
U.S. Pat. No. 3,976,451 describes selectively permeable membranes for passing oxygen.
U.S. Pat. No. 4,260,392 shows a selectively permeable plastic tape.
U.S. Pat. No. 3,897,214 discloses reagents impregnated in plastic fibers.
U.S. Pat. No. 3,697,266 discloses a system using a graded scale for visual comparison. The comparison scale is not placed in a solution. It is merely a screen.
U.S. Pat. No. 998,091 discloses a color comparing scheme in which thickness is varied in a graded standard.
U.S. Pat. Nos. 4,181,501 and 4,054,490 disclose wedge shaped concentration sensors.
U.S. Pat. No. 2,351,644 discloses a stepped sensor.
U.S. Pat. No. 4,073,623 discloses a non-immersed sensor and standard used for visual comparisons.
U.S. Pat. No. 1,456,964 discloses light intensity comparison.
U.S. Pat. No. 4,003,707, Lubbers et al, discloses a method and apparatus for detecting oxygen wherein the detecting material is incorporated in a matrix which is permeable to oxygen but impermeable to interfering materials. Lubbers et al further discloses that the detecting material or indicator can be embedded in a foil which serves as a gas permeable membrane and a light transmissive surface by polymerization of a solution of silicone or any synthetic plastic material, such as polyvinylchloride mixed with the indicating substance. Lubbers et al also discloses that the device may comprise a plurality of small carrier particules having the indicating substance embedded therein. Lubbers et al has a membrane which separates the reactive material in a cell from the substance being tested. Lubbers et al teaches a harsh method not amenable to more modern sensitive indicators. Reversible phosphorescing complexes are sensitive indicators, especially heavy metal atom, stabilized phosphorescing complexes wherein emissions specifically involve the metal ion. Polymerizing these complexes into selectively permeable matrices is out of the question. Methods are needed to immobilize and embed these modern complexes without physically or chemically altering their structures.
U.S. Pat. No. 3,904,373, Harper, discloses the concept of binding indicators to insoluble carriers with covalent bonding. The indicators specifically mentioned are organic chemicals. The insoluble carriers are glass mixtures. The reaction to bind the indicators needs a silicone coupling agent, refluxing and distillation apparatuses. Sensitivities of more modern indicators, such as heavy metal, atom stabilized phosphorescing complexes preclude the use of such teachings. Moreover, glass is not an optimal matrix in light of more modern insoluble carriers. What is needed is a simple diffusion method of immobilizing an indicator substance in an insoluble matrix such as silicone rubber. Such a matrix would be more versatile and amenable to a plethora of probe embodiments.
U.S. Pat. No. 4,255,053, Lubbers et al, discloses a method and apparatus for an optical measurement of the concentration of substances wherein the device includes a reference device with various concentrations of a reference material. Improvements are needed in this method because the sensor and reference are not aligned in proximate relationship. Using various calibration ranges is not a contemporaneous measurement and is cumbersome. Microscopic particles used in a matrix is suggested but single isolated particles are needed for monitoring [O.sub.2 ] under a microscope for lifetime quenching methods. Moreover, Lubbers specifically excludes using the same material for the reference and the sensor. Column 2, lines 59-64. Furthermore, a detailed analysis of the equations shows that the design cannot be made to work with a reference that has an emission identical to reversible phosphorescing material. This is a consequence of the fact that Lubbers must separate the signals from these materials optically by wavelength while what is needed is a separation done by using either two detectors or by time multiplexing the two signals onto one detector. While Lubbers suggests using several reference indicators (column 5, 15-7), the exclusion of using the same indicator substance for reference and sensor completely overlooks the enormous advantages. These advantages include: (1) By comparing identical colors on both channels one avoids any ambiguities in the spectral sensitivity of the detector, especially if the same detector is time multiplexed between the two samples; (2) Use of the same material in both channels makes the responses largely temperature independent if they are thermally lagged to each other since the intensity of both will rise and fall together as the temperature is varied.
USSR Patent 893,853, Leningrad, discloses a method for detecting oxygen wherein the oxygen is detected by the quenching of a luminescent material. This reference describes an indicator within a silica gel. The silica gel is a highly absorbent drying agent which would draw deoxygenated water as well as residual oxygen into the silica gel. What is needed is a carrier that is gas permeable and solvent impermeable.
The remaining patents are of lesser interest.
The following publications are also of interest.
Energy Transfer in Chemiluminescence, Roswell, Paul and White, Journal of the American Chemical Society, 92:16, Aug. 12, 1970, pp. 4855-60; Oxygen Quenching of Charge-Transfer Excited States of Ruthenium (II) Complexes. Evidence for Singlet Oxygen Production, Demas, Diemente and Harris, Journal of the American Chemical Society, 95:20, Oct. 3, 1973, pp. 6864-65; Energy Transfer from Luminescent Transition Metal Complexes to Oxygen, Demas, Harris and McBride, Journal of the American Chemical Society, 99:11, May 25, 1977, pp. 3547-3551; Britton, Hydrogen Ions. Their Determination and Importance in Pure and Industrial Chemistry, D. Van Nostrand Company, Inc. (1943) pp. 338-43; and Fiberoptics Simplify Remote Analyses, C&EN, Sept. 27, 1982, pp. 28-30. Porphyrins XVIII. Luminescence of (Co), (Ni), Pd, Pt Complexes, Eastwood and Gouterman, Journal of Molecular Spectroscopy, 35:3, Sept. 1970, pp. 359-375; Porphrins. XIX. Tripdoublet and Quartet Luminescence in Cu and VO Complexes, Gouterman, Mothies, Smith and Caughey, Journal of Chemical Physics, 52:7, Apr. 1, 1970, pp. 3795-3802; Electron-Transfer Quenching of the Luminescent Excited State of Octachlorodirhenate (III), Nocera and Gray, Journal of the American Chemical Society 103, 1971, pp. 7349-7350; Spectroscopic Properties and Redox Chemistry of the Phosphorescent State of Pt.sub.2 (P.sub.2 O.sub.5).sub.4 H.sub.8.sup.4-, Che, Butler and Gray, Journal of the American Chemical Society 103, 1981, pp. 7796-7797; Electronic Spectroscopy of Diphosphine- and Diarsine-Bridget Rhodium (I) Dimers, Fordyce and Crosby, Journal of the American Chemical Society 104, 1982, pp. 985-988.
The Demas et al articles disclose oxygen quenching of .alpha.-diimine complexes of Ru(II), Os(II), and Ir(III). 2, 2'-bipyridine, 1,10-phenanthroline and substituted derivatives are used as ligands to form the metal-ligand complexes. A kinetic mechanism for the complex oxygen interaction is proposed.
The Roswell article discusses intermolecular energy transfer in chemiluminescence.
The Britton publication discloses a wedge method for the determination of indicator constants of two-color indicators.
The C&EN article deals with PTFE control membranes in the context of laser optodes and optical fibers.
The Eastwood article describes the room temperature luminescence and oxygen quenching of Pd and Pt porphyrin complexes in fluid solutions.
The Gouterman et al article describes low temperature luminescence of Cu and VO porphyrins. Extrapolation of their data to room temperature indicates oxygen quenchable lifetimes.
The Nocera paper reports quenching of dinuclear Re species. Mononuclear and dinuclear Re complexes also have quenchable excited states.
The Che paper reports long excited state lifetimes and solution oxygen quenching of a dimeric Pt complex in solution and long-lived quenchable excited states of Rh dimers.
The Fordyce reference reports long-lived low temperature emissions of Rh(I) with bridging ligands. Rh(I) and Ir(I) data are referenced. Extrapolation of their data to room temperature suggests oxygen quenchable lifetimes.
A major problem with existing sensor materials is that they are organic molecules that generally suffer from instabilities and/or short fluorescence lifetimes, which make them relatively insensitive to oxygen quenching. While, in principle, there are long-lived phosphorescences, the extant molecules at the time of the earlier patents were all unstable and therefore unsuitable as sensors. Indeed, Stanley and Kropp (U.S. Pat. No. 3,725,648, col. 2, 1 46-50) explicitly excluded phosphorescent molecules as potential sensor materials because of the stability problems. Lubbers (U.S. Pat. No. 4,255,053 and 4,003,707) never goes on to correct (extend, change) this limitation. Therefore, new classes of more stable long-lived sensor materials are required.
A further problem with existing sensor materials is the preparation of a suitable film with the luminescent sensor. One preparation procedure evaporates the films from a solution containing polymer and luminescent sensor material; many polymers (e.g. silicones) cannot be evaporated since they are not soluble in suitable solvents. The other suggested procedure is to polymerize the luminescent sensor material with the monomer. However, most polymerizations are free radical processes that can destroy much or all of the luminescent material and create undesirable side products.
Another problem with existing technologies is that they use luminescence intensity to measure the degree of interaction with the quencher: Since the emission intensity is a function of the temperature of the film, any fluctuation in the temperature of the sensor will appear as apparent changes in the quencher concentration. A further problem is that unless a double beam arrangement is used, excitation source fluctuations appear as erroneous concentration changes.
An additional disadvantage of existing technologies is that there is no inexpensive visual method for monitoring gaseous concentrations of various species. Also, the methods of protecting sensors from the environment are limited. Furthermore, no method is suitable for measuring concentrations of dissolved gases such as oxygen in such systems as growing cells under a microscope or in the central core of a flow cytometer cell where any invasive device such as a catheter would cause the system to stop functioning.
All of the systems in the art either lack stability, suffer from a variety of interferents, lack enabling details to implement, or are merely concepts rather than working models.